There is currently an unmet clinical need for diagnostic tests that rapidly provide direct evidence and localization of foreign-body infections (e.g., catheter and prosthetic-joint infections). Staphylococcus aureus (S. aureus) is the second most common bacterial species responsible for foreign-body infections and it is responsible for much of the morbidity and mortality that result from such infections due to its highly virulent nature. This proposal is based on a novel molecular imaging approach that rapidly and specifically detects S. aureus infections with a fluorescent probe that is activated by an S. aureus-specific nuclease. The long-term goal of this line of investigation is to translate this approach into clinical practice. The objective of this application is to carry out preclinical evaluation of an optimized S. aureus-specific activatable probe in a murine foreign-body S. aureus infection model. The central hypothesis of this proposal is that an optimized probe will exhibit low toxicity and high sensitivity and specificity for imaging S. aureus foreign-body infections in mice. The Specific Aims are: 1) Identify a Next Generation TT probe (NGTT probe) by incorporating a fluorophore/quencher combination that is optimal for in vivo imaging. The spectral characteristics and activated/unactivated fluorescence ratio of the first generation TT probe will be optimized with the design, generation and comparison of several probe variants with red-shifted fluorophores and appropriately matched quenchers. These probes will be evaluated in vitro and in a pyomyositis S. aureus infection model. The best- performing probe will be selected as the NGTT probe. 2) Image S. aureus foreign-body biofilm infections in mice with the NGTT probe. Foreign-body biofilm infections are a large and problematic category of S. aureus infections. The utility of the NGTT probe will be tested in a mouse model of such infections in which S. aureus biofilms grow on catheters implanted in the mice. 3) Assess the safety of the NGTT probe in mice. Various measures of toxicity, including complete blood counts will be taken in mice after intravenous administration of the NGTT probe. Doses of the probe, 10-fold greater than those found to yield robust infection imaging, will be administered. The proposed work is expected to result in the optimization and thorough pre-clinical testing of this approach for imaging S. aureus foreign-body biofilm infections. This contribution is significant because it will provide an essential foundation for the translation of a diagnostic imaging approach for S. aureus infections, and thus constitutes critical progress towards addressing an unmet clinical need. The work proposed is novel in several respects. This will be the first thorough preclinical assessment of a nuclease-activated oligonucleotide probe for the non-invasive diagnostic imaging of any medical condition. This work also represents the first evaluation of efficacy and specificity of our imaging approach in a model of a common clinical problem, an important step towards clinical translation. Finally, the application of this approach to foreign-body biofilm infections constitutes an innovative solution to a challenging problem.